Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Introduction
Cardiovascular disease is one of the leading causes of death in the world.
Formation of harmful reactive oxygen species (ROS) is associated with
several pathological conditions, and contributes to ischaemia/reperfusion
injury. Antioxidants can be added to the diet in an attempt to decrease the
prevalence of cardiovascular disease by decreasing the harmful effects of
ischaemia/reperfusion injury.
Red Palm Oil (RPO) consists of saturated, monounsaturated and
polyunsaturated fatty acids and is rich in antioxidants such as -carotene,
tocopherols and tocotrienols.
It has previously been shown that RPO-supplementation improved
reperfusion mechanical function. In these studies it was found that RPO
might exert its beneficial effects during reperfusion through increased PKB/Akt
pathway activity, which may lead to inhibition of apoptosis and improved
mechanical function.
Aims
The aims of this study were: 1) to determine whether RPO-supplementation
protected against ischaemia/reperfusion injury in the isolated perfused rat
heart, 2) to confirm RPO-supplementation’s effect on the PKB/Akt pathway
activity and, 3) to elucidate the regulators in the PKB/Akt pathway that RPOsupplementation
influenced.
Methods
Male Wistar rats were divided into 4 groups, 2 control groups and 2
experimental groups. The 2 control groups were fed a standard rat chow
(SRC) for 4 weeks. The two experimental groups received SRC and RPOsupplementation
for 4 weeks. Hearts were excised and transferred to a
Langendorff perfusion apparatus and perfused with Krebs-Henseleit buffer. Mechanical functional recovery was measured after 25 min of total global noflow
ischaemia. The following parameters were also measured during various
time points in the protocol: left ventricular develop pressure, heart rate,
coronary flow, rate pressure product. Hearts were also freeze-clamped for
biochemical analysis at 10 min during reperfusion. The biochemical analysis
was aimed at determining PKB/Akt involvement.
In a second protocol, hearts were subjected to the same perfusion protocol,
but wortmannin was also added to the perfusion fluid, in order to inhibit PI3-
kinase.
Results
Hearts from the RPO-supplemented rats showed an improved RPP recovery
(92.26 ± 5.89 % vs 63.86 ± 7.74 %) after 10 min of reperfusion. This finding
corroborated the findings of previous studies. Hearts of the RPOsupplemented
rats perfused with wortmannin, showed increased RPP
recoveries at several time points.
Biochemical results showed that wortmannin did indeed inhibit PI3-K
phosphorylation in the RPO-supplemented group, as was expected. The
RPO-supplemented group that was perfused with wortmannin had an
increased PKB/Akt (Ser473) phosphoyrylation, when compared to the
wortmannin control group. It was also found that the combination of RPO and
wortmannin had prosurvival effects.
Discussion
This study showed that RPO-supplementation offered protection against
ischaemia/reperfusion injury in the Langendorff-perfusion apparatus at 10 min
into reperfusion. Thereafter the significance of the protection was lost. This
protection has been confirmed in several previous studies and several
mechanisms have been proposed for this protection.
Since no conclusive evidence exists on the precise mechanism of protection,
our investigation focused on the regulators of the pro-survival PKB/Akt
pathway. An improved functional recovery was also seen in the RPO-supplemented
group that was perfused with wortmannin. This was an unexpected finding,
because Wortmannin is a known PI3-kinase inhibitor (as was confirmed by
our biochemical data). PI3-kinase phosphorylation leads to PKB/Akt
phosphorylation and therefore, activation of a pro-survival pathway. It would
be expected that wortmannin would inhibit PKB/Akt and thus decrease the
survival of the cells. The RPO-supplementation thus reversed wortmannin’s
detrimental effect to such an extent that the functional recovery was far better
than RPO-supplementation alone.
In the RPO + wortmannin group, PKB/Akt (Ser473) phosphorylation was
increased, contrary to previous findings. This is an indication that RPO may
have the ability to override wortmannin’s inhibitory effect on PI3-kinase, or
that PKB/Akt (Ser473) may be phosphorylated independently of PI3-kinase. / AFRIKAANSE OPSOMMING: Inleiding
Kardiovaskulêre siektes is een van die hoof oorsake van sterftes in die
wêreld. Die vorming van skadelike reaktiewe suurstof spesies word
geassosieer met verskeie patologiese kondisies en dra ook by tot
isgemie/reperfusie skade. ‘n Moontlike manier om die voorkoms van
isgemie/herperfusie skade asook kardiovaskulêre siektes te voorkom, is om
antioksidante by die dieet te voeg.
Rooi Palm Olie (RPO) bevat versadigde, mono-onversadigde en polionversadigde
vetsure. RPO bevat ook ‘n oorvloed van antioksidante soos β-
karoteen en tokoferole en tokotriënole.
Dit is bewys in vorige studies dat RPO-aanvulling verbeter funksionele
herstel. Hierdie voordelige effekte mag dalk wees agv verhoogde PKB/Akt
pad aktiwiteit. Die PKB/Akt pad word geassosieer met die inhibisie van
apoptose en verhoogde meganiese funksie.
Doelwitte
Die doelwitte van hierdie studie was om te bepaal of 1) RPO-aanvulling
beskermende effekte teen isgemie/herperfusie skade in die geisoleerde
rotharte het, 2) Bevestig of RPO-aanvulling wel die PKB/Akt pad beïnvloed 3).
om die effekte wat RPO-aanvulling het op die reguleerders van die PKB/Akt
pad te onthul.
Metodes
Manlike Wistar rotte is in 4 groepe verdeel. 2 Groepe kontrole rotte is ‘n
standaard rotkosmengsel gevoer vir 4 weke. Die 2 eksperimentele groepe
het ook ‘n standaard rotkosmengsel gekry plus ‘n RPO-aanvulling vir 4 weke.
Harte is uitgesny en op ‘n Langendorff perfusie sisteem gemonteer en met
Krebs-Henseleit buffer geperfuseer. Meganiese funksie herstel is gemeet na
25 min totale globale geen-vloei isgemie. Linker ventrikulêre ontwikkelde
druk, harttempo, koronêre vloei en tempo druk produk is gemeet by verskillende tydpunte. Sommige harte is na 10 min herperfusie vir
biochemiese analiese gevriesklamp. Die biochemiese analisiese was beoog
om die PKB/Akt pad betrokkenheid te bepaal.
‘n Tweede stel harte is aan dieselfde perfusie protokol blootgestel, maar
wortmannin (PI3-kinase inhibitor) is ook bygevoeg by die perfusie vloeistof.
Resultate
Die groep wat met RPO aangevul is, het na 10 min herperfusie, ‘n verbeterde
tempo druk produk herstel getoon (92.26 ± 5.89 % vs 63.86 ± 7.74. Hierdie
bevinding is ook met ander studies bevestig. ‘n Interessante bevinding was
dat die groep wat met RPO aangevul is en met wortmannin geperfuseer is, ‘n
verbeterde meganiese funksionele herstel getoon het.
Biochemiese resultate het getoon dat wortmannin wel PI3-K fosforilering
geinhibeer het. Die harte van die rotte in die groep wat aangevul is met RPO
en daarna met wortmannin geperfuseer is, het ‘n toename in PKB/Akt (Ser473)
fosforilering getoon, relatief tot die wortmannin geperfuseerde harte van die
rotte in die kontrole groep. Hierdie groep (RPO-aanvulling en wortmannin
perfusie) het beskermende effekte getoon.
Bespreking
Hierdie studie het getoon dat RPO-aanvulling beskerming gebied het teen
isgemie/herperfusie skade in die Langendorff geperfuseerde rothart na 10 min
herperfusie. Daarna is die beduidenheid van die beskerming verloor. Hierdie
bevindings ondersteun die resultate van vorige studies. Verskeie moontlike
meganismes is voorgestel vir die beskerming, maar die presiese meganisme
is nog nie duidelik nie.
In hierdie studie is daar gekyk na die reguleerders van die PKB/Akt pad.
Geen vorige studies het al gefokus op RPO-aanvulling en sy effek op die
reguleerders van die PKB/Akt pad nie.
‘n Onverwagte bevinding is dat harte van die rotte in die RPO + wortmannin
groep ‘n verbeterde funksionele herstel getoon het. Wortmannin is ‘n PI3- kinase inhibitor. PI3-K fosforilering lei tot PKB/Akt fosforilering, wat tot sel
beskerming lei. Dus, aangesien wortmannin PI3-K inhibeer, sou dit verwag
word dat wortmannin sel beskerming sal verminder. Die RPO het egter die
wortmannin se nadelige effekte tot so ‘n mate oorskrei dat die funksionele
herstel baie beter was as die RPO-aanvulling alleen.
Die verhoogde PKB/Akt (Ser473) fosforilering, wat gesien is in die RPO +
wortmannin groep kan toegeskryf word aan RPO se vermoë om wortmannin
se nadelige effekte te oorskrei. ‘n Moontlike verduideliking vir hierdie
bevinding mag wees dat rooi palm olie PKB/Akt (Ser473) op ‘n PI3-K
onafhanklike manier fosforileer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21745 |
| Date | 12 1900 |
| Creators | Odendaal, Louise |
| Contributors | Engelbrecht, A.M., Van Rooyen, J., Du Toit, E.F., Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xx, 98 leaves : ill. |
| Rights | Stellenbosch University |
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